Glioblastoma multiforme (GBM) is a devastating disease with a median survival of only about a year. Little progress has been made in the treatment of GBM during the last 30 years. Thus, novel approaches and therapeutic agents are urgently needed. We recently showed that the second-generation ATM inhibitor (ATMi), KU-60019, is a potent radiosensitizer in the first report published on this novel compound. Briefly, KU-60019 is a very specific ATM kinase inhibitor in vitro and impressively radiosensitizes human glioma cells irrespective of PTEN and p53 status but has much better effect in vivo with mutant p53 gliomas. In addition, our studies have shown that glioma cell migration and invasion in vitro were inhibited by KU-60019 perhaps by interfering with AKT and ERK signaling. Thus, the potential clinical benefit of an ATMi as a radiosensitizer for GBM is not limited to its abilit to block the DNA damage response (DDR) and potently radiosensitize glioma cells but also to inhibit invasion and spread of the cancer in between radiation fractions. However, one of the drawbacks with KU-60019 is that it does not cross the blood-brain-barrier (BBB) and requires direct intra-tumoral delivery to be effective. Thus, an ATMi able to cross the BBB would facilitate and improve treatment at every level. Herein, we will test a third-generation ATMi, AZ31, with enhanced properties for treating brain tumors. Our preliminary data shows that AZ31 radiosensitizes human and mouse glioma cells and blocks the DDR in vitro, and prolongs the survival of mice growing syngeneic orthotopic tumors with mutant p53. However, radiation scheduling and dosing using a representative panel of human xenografts, such as glioma stem cells, now needs to be performed in order to bring this compound closer to clinical testing. Thus, further testing of AZ31 in mouse glioma models using clinically relevant human GBM xenografts with different p53 status as well as other critical GBM characteristics is now warranted (Aim 1). Little is known about the molecular processes occurring in the brain in response to DNA damage resulting from an ATMi and radiation but, in general, it is believed that neurogenesis would be impaired due to DDR sequelae and inflammation. Except for stem cells and neural progenitors (NPs), the brain consists mostly of terminally differentiated cells that do not proliferate. Thus, aggressively growing glial brain tumors residing in the brain parenchyma would be very favorable for therapeutic intervention with AZ31, in particular if the tumor carries mutation in p53, which is the case at least 30% of the time. In this case one expects the therapeutic gain to be highly favorable. Nevertheless, it is very important to examine what effect this treatment might have on the neural compartment so that appropriate steps can be taken to spare normal tissue such as using conformal radiation (Aim 2). We expect that insights gained from the proposed animal studies will demonstrate proof-of-principle of a novel `synthetical lethal' drug and radiation combination strategy for the treatment of GBM that would be effective and safe.